Investigation of genetic variations in the mARC-containing N-reductive enzyme system

Das mARC-haltige N-reduktive Enzymsystem besteht aus mARC-, Cytochrom b5 Typ B und NADH Cytochrom b5 Reduktase und ist in der Lage, N-hydroxylierte Verbindungen in Gegenwart von NADH zu reduzieren. Ziel dieser Arbeit war es, 12 zum Aminosäureaustausch führende SNPs in diesem Enzymsystem zu untersuchen. Vier SNPs in MARC1 (c.493A>G, c.560T>A, c.736T>A und c.739G>C) und zwei SNPs in MARC2 (c.730G>A und c.735T>G) wurden mittels Pyrosequencing genotypisiert. Sie wiesen insgesamt nur eine geringe Frequenzhäufigkeit in der untersuchten Kohorte von 340 gesunden Kaukasiern auf. Nur für einen SNP in MARC1 (c.493A>G) konnte die homozygote Variante mit einer Frequenz von 7,1% detektiert weden. Dieser SNP führte zur Proteinvariante A165T. Die Proteinvarianten von mARC (mARC-1: V96L, A165T, M187K, C246S, D247H und M268I; mARC-2: G244S und C245W) und Cytochrom b5 Typ B (S2F, D14G, K16E und T22A) wurden in E. coli exprimiert und mittels in vitro Biotransformations-studien am Substrat Benzamidoxim, dem Modellsubstrat der Amidoximprodrugs, untersucht. Die katalytische Effizienz der Varianten G244S und C245W war im Vergleich zu ihrem Wildtypprotein signifikant erniedrigt, ebenso die des mARC-2-Wildtypproteins im Vergleich zum mARC-1-Wildtypprotein. Die mARC-1-Variante A165T und alle Cytochrom b5 Typ B-Varianten beeinflussten die N-reduktive Aktivität des Enzymsystems nicht. Die mARC-1-Mehrfachvariante, in der alle sechs Aminosäuren (V96L, A165T, M187K, C246S, D247H und M268I) ausgetauscht waren, zeigte trotz Bindung des Molybdäncofaktors nahezu keine N-reduktive Aktivität mehr. Dies könnte ein erster Hinweis auf mögliche Substratbindestellen sein. Polymorphismen können zu Arzneimittelnebenwirkungen führen. Die Beteiligung des N-reduktiven Enzymsystems an der Reduktion von Sulfamethoxazolhydroxylamin wurde in vitro gezeigt. Die mARC- und Cytochrom b5 Typ B-Proteinvarianten beeinflussten die Reduktion nicht, so dass eine Beteiligung an der Pathogenese der Hypersensitivitätsnebenwirkung unter Sulfamethoxazol sehr unwahrscheinlich ist. mARC-1 besaß die höhere katalytische Effizienz in der Reduktion von Benzamidoxim und mARC-2 in der Reduktion von Sulfamethoxazolhydroxylamin. Erste Versuche zur Etablierung eines Biomarkertests zu einer Nebenwirkung des Amidoxim-Prodrugs Ximelagatran, die von der Firma caprotecTM bioanalytics GmbH durchgeführt wurden, zeigten eine Wechselwirkung zwischen der Melagatran Capture Compound und dem mit dem löslichen HLA-Molekül DRB1*0701.Mitochondrial amidoxime reducing component (mARC), cytochrome b5 type B and NADH cytochrome b5 reductase form an N-reductive enzyme system that is capable of reducing N-hydroxylated compounds in the presence of NADH. Aim of this work was to investigate 12 nonsynonymous SNPs located in this enzyme system. Genotype frequencies of four SNPs in MARC1 (c.493A>G, c.560T>A, c.736T>A and c.739G>C) and two in MARC2 (c.730G>A and c.735T>G) were investigated by pyrosequencing. The six variants were of low frequency in a cohort of 340 healthy Caucasian. Only one homozygous variant (493AA, MARC1) was detected with a frequency of 7.1% leading to the protein variant A165T. Protein variants of mARC (mARC-1: V96L, A165T, M187K, C246S, D247H and M268I; mARC-2: G244S and C245W) and cytochrome b5 type B (S2F, D14G, K16E and T22A) were expressed in E. coli. In vitro biotransformation studies were performed with benzamidoxime, the model compound for amidoxime prodrugs. Catalytic efficiencies of mARC-2 variants G244S and C245W were significantly decreased compared with their wild type protein. Catalytic efficiency of mARC-1 wild type protein was significantly decreased compared with mARC-2 wild type protein. mARC-1 variant A165T and cytochrome b5 type B variants S2F, D14G, K16E and T22A did not influence the N-reductive activity. After replacement of all six amino acids (V96L, A165T, M187K, C246S, D247H and M268I) in one mARC-1 protein N-reductive activity was almost absent. Possibly these residues are important for substrate binding altogether. Genetic polymorphisms are able to cause adverse drug reactions. Participation of the N-reductive enzyme system in the reduction of sulfamethoxazole hydroxylamine was demonstrated in vitro. Protein variants of mARC and cytochrome b5 type B did not influence this N-reduction and most likely would not participate in the pathogenesis of hypersensitivity reactions after treatment with sulfamethoxazole. When catalytic efficiencies for both substrates were compared, mARC-1 seemed to be more efficient in the N-reduction of benzamidoxime. mARC-2 was more efficient in the N-reduction of sulfamethoxazole hydroxylamine. Initial experiments focusing on the establishment of a biomarker test that might be able to detect patients at risk of adverse reactions after treatment with ximelagatran were performed by the company caprotecTM bioanalytics GmbH in Berlin. A first interaction between a melagatran capture compound and the soluble MHC class II molecule DRB1*0701 was shown

Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

Background: The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results: Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions: Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions

S1 Guideline onychomycosis

Onychomycosis is a fungal infection of the fingernails and toenails. In Europe, tinea unguium is mainly caused by dermatophytes. The diagnostic workup comprises microscopic examination, culture and/or molecular testing (nail scrapings). Local treatment with antifungal nail polish is recommended for mild or moderate nail infections. In case of moderate to severe onychomycosis, oral treatment is recommended (in the absence of contraindications). Treatment should consist of topical and systemic agents. The aim of this update of the German S1 guideline is to simplify the selection and implementation of appropriate diagnostics and treatment. The guideline was based on current international guidelines and the results of a literature review conducted by the experts of the guideline committee. This multidisciplinary committee consisted of representatives from the German Society of Dermatology (DDG), the German‐Speaking Mycological Society (DMykG), the Association of German Dermatologists (BVDD), the German Society for Hygiene and Microbiology (DGHM), the German Society of Pediatric and Adolescent Medicine (DGKJ), the Working Group for Pediatric Dermatology (APD) and the German Society for Pediatric Infectious Diseases (DGPI). The Division of Evidence‐based Medicine (dEBM) provided methodological assistance. The guideline was approved by the participating medical societies following a comprehensive internal and external review

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, Km and kcat, are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions

Der Klassenrat als Chance für Partizipation

Grüning M., Martschinke S., Häbig J. & Ertl S. (Hrsg). Mitbestimmung von Kindern. Grundlagen für Unterricht, Schule und Hochschule. Beltz Juventa, S. 193-212

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are dynamic light scattering (DLS), UV-Vis spectrophotometry and isothermal titration calorimetry (ITC) respectively. The workflow is standardized by the use of SOPs and python-scripted data analysis. We have used the NADPH-dependent alcohol dehydrogenase Gre2p as a challenging enzyme to demonstrate the power of this workflow. Our work highlights the utility for combined binding and kinetic studies for such complex multi-substrate reactions and the importance of sample quality control during experiments. </div